Presence Of Inducer Research Articles

Extracellular arabinases in Aspergillus nidulans: the effect of different cre mutations on enzyme levels.

The regulation of the syntheses of two arabinan-degrading extracellular enzymes and several intracellular L-arabinose catabolic enzymes was examined in wild-type and carbon catabolite derepressed mutants of Aspergillus nidulans. alpha-L-Arabinofuranosidase B, endoarabinase, L-arabinose reductase, L-arabitol dehydrogenase, xylitol dehydrogenase, and L-xylulose reductase were all inducible to varying degrees by L-arabinose and L-arabitol and subject to carbon catabolite repression by D-glucose. With the exception of L-xylulose reductase, all were clearly under the control of creA, a negative-acting wide domain regulatory gene mediating carbon catabolite repression. Measurements of intracellular enzyme activities and of intracellular concentrations of arabitol and xylitol in mycelia grown on D-glucose in the presence of inducer indicated that carbon catabolite repression diminishes, but does not prevent uptake of inducer. Mutations in creA resulted in an apparently, in some instances very marked, elevated inducibility, perhaps reflecting an element of "self" catabolite repression by the inducing substrate. creA mutations also resulted in carbon catabolite derepression to varying degrees. The regulatory effects of a mutation in creB and in creC, two genes whose roles are unclear, but likely to be indirect, were, when observable, more modest. As with previous data showing the effect of creA mutations on structural gene expression, there were striking instances of phenotypic variation amongst creA mutant alleles and this variation followed no discernible pattern, i.e. it was non-hierarchical. This further supports molecular data obtained elsewhere, indicating a direct role for creA in regulating structural gene expression, and extends the range of activities under creA control.

Induction of laccase production inCyathus bulleri under shaking and static culture conditions

Laccase production byCyathus bulleri was lower in lignins and phenolic compounds as compared to malt extract medium (8 U/mL) which increased significantly on supplementing these compounds with malt extract. Of the different lignins and phenolic compounds, Reax, lignin and orcinol exhibited maximum laccase formation (12 and 68 U/mL, respectively) under static culture conditions, while sugars repressed it. Laccase activity inC. bulleri was higher under static than under shaking cultivation conditions. Moreover, agitation repressed laccase formation even in the presence of inducers.

Loss of glucose transporters is an early event in differentiation of HD3 cells.

The HD3 cell, a chicken erythroblast cell line infected with a temperature-sensitive avian erythroblastosis virus, becomes committed to differentiate to an erythrocyte upon temperature shift in presence of inducers. Before induction, the HD3 cell transports glucose and 2-deoxyglucose (2-DG). 3-O-methylglucose is poorly taken up. Upon induction of differentiation, glucose and 2-DG transport activity fall. Twenty-four hours postinduction, up to 75% of the glucose transport activity may disappear. By use of cDNA probes for chicken glucose transporters, two species of mRNA of 3.1 and 1.7 kb (equivalent to mammalian GLUT1 and GLUT3 mRNA, respectively) are detected. Both messages virtually disappear within 48 h after induction. Run-on assays show the cessation of synthesis of the corresponding RNAs parallel to the loss of glucose transport. In contrast to the glucose transporters, the nucleoside transporter level increases after induction of hematopoiesis. This developmental pattern is consistent with earlier studies showing that mature chicken erythrocytes have little glucose transport activity but retain appreciable levels of the nucleoside transporter and that nucleosides and glutamine provide major sources of oxidizable carbon compounds to sustain metabolism in circulating chicken erythrocytes.

Crotonobetaine reductase fromEscherichia coli ? a new inducible enzyme of anaerobic metabolization of L(-)-carnitine

Crotonobetaine reductase from Escherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, gamma-butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, gamma-butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40-45 degrees C). The Km value for crotonobetaine was determined to be 1.1 x 10(-2M). gamma-Butyrobetaine, D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For gamma-butyrobetaine (Ki = 3 x 10(-5M)) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.

Utilization of a mim-D lac transposable element to create an α-complementation and regulated expression system for cloning in Pseudomonas aeruginosa

A lac-based α-complementation and expression system was developed for use in molecular cloning in Pseudomonas aeruginosa. A bacteriophage D3112-based mini-D lac transposable element, containing the lacI q-regulated lacZΔM15 gene next to a selectable marker, was constructed. Mixed D3112 lysates were used to transduce P. aeruginosa PAO1, and derivatives containing randomly inserted chromosomal copies of the mini-D lac element were obtained. Transformation of the PAO1::mini-D lac transductants with the broad-host-range vector, pUCP19, led to the formation of blue colonies on indicator medium m the presence of inducer. In contrast, transformants harboring the pUCP19 derivative pCDO, containing the catechol-2,3-dioxygenase (C230)-encoding xylE gene under lac promoter control, were white on the same medium. Expression of xylE was tightly controlled by single-copy mini-D lac-encoded lac repressor and in induced cultures was increased more than 100-fold over that observed in uninduced cultures. The usefulness of the system for molecular cloning in P. aeruginosa was demonstrated by ligating size-fractionated PAO1 chromosomal fragments into pUCP19, followed by transformation of the newly isolated PAO1::mini-D lac host. All randomly chosen white colonies contained recombinant plasmids, with inserts of the correct size range, while blue colonies contained pUCP19 alone. The functionality of the system was also shown in another frequently studied strain, PA103.

2,4-D degradation in monoculture biofilm reactors

Two strains of Alcaligenes eutrophus AEO106 were used to inoculate expanded bed biofilm reactors which were exposed to a range of concentrations and loading rates of 2,4-dichlorophenoxyacetic acid (2,4-D herbicide). Two bench-scale reactors were inoculated with bacterial strains containing different plasmids. Plasmid pRO101 expressed the 2,4-D degradative pathway in the presence of inducer (2,4-D or 3-chlorobenzoate), whereas plasmid pRO103 was engineered to allow constitutive expression of the pathway due to the deletion of a regulatory gene. Biofilm growth characteristics, loading rate and 2,4-D conversion efficiency relationships were compared in the two reactors. Actual loading rates tested ranged from 3.6 to 52.3 g 2,4-D per liter expanded bed volume per day (g/ld). The range of influent concentrations tested was between 166 and 728 mg/l (0.017 and 0.078%). Mineral nutrients were supplied in the feed solutions. At loadings up to 15 g/ld, reactors were aerated with filtered air. Pure oxygen was found to be necessary at higher loadings to sustain maximum bacterial activity. Removal of 2,4-D to below detectable concentrations (< 3 mg/l) was demonstrated at all but the highest loadings tested (at least up to 10 g/ld with air and > 25 g/ld with pure oxygen). However, minimum effluent chemical oxygen demand (COD) values were 25 mg/l even at lower loading conditions. Advantages noted with the AEO106 pRO103 (constitutive) biofilm included greater biomass development (up to 3 times higher volatile solids), 50–100% higher specific substrate conversion rates, quicker response to step increases in feed rate and concentration and more consistent effluent quality. Approximately one-third lower effluent COD concentrations, with one-half the standard deviation among individual analyses, were noted with pRO103 compared to pRO101. The genetically engineered biofilm reactor was shown to be a viable experimental treatment technology for one xenobiotic compound in these studies. However, pure culture conditions were not maintained throughout the experiments because of the low-level persistence of a sporadic contaminant bacterial species (identified as Xanthomonas maltophilia), which apparently incorporated plasmids from Alcaligenes eutrophus AEO106.

Changes in cytoplasmic pH upon heat shock in embryonic and adult rat liver ceils

All living cells, when exposed to elevated temperatures, undergo physiological changes which result in the expression of a specific set of heat shock proteins. Study of the possible physiological changes in adult and embryonic rat liver cells indicated a change in intracellular pH upon heat shock. Using 2′, 7′-bis (2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethyl ester, we demonstrate here that the intracellular pH of adult and embryonic liver cells is different and that there is an increase in relative fluorescence intensity in both adult and embryonic cells upon heat shock, which corresponds to about 0.2 to 0.3 pH units. We also show that in addition to heat, some of the inducers of heat shock like response in many systems also induce a change in intracellular pH and induce heat shock proteins at 37°C in fetal liver cells. The possible mechanisms of induction of heat shock proteins during heat shock and in the presence of inducers at normal temperature are discussed.

The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli.

Most genes required for cysteine biosynthesis in Salmonella typhimurium and Escherichia coli are positively regulated by cysB, which encodes a transcriptional activator belonging to the LysR family of regulatory proteins. CysB protein binds just upstream of the -35 region of positively regulated promoters, where in the presence of inducer it facilitates formation of a transcription initiation complex. CysB protein also autoregulates its own synthesis by binding to the cysB promoter as a repressor. Cysteine down-regulates the pathway by inhibiting synthesis of O-acetylserine, a direct cysteine precursor and possibly an inducer of gene expression. O-Acetylserine spontaneously isomerizes to N-acetylserine, which is clearly an inducer. Sulphide and thiosulphate provide additional regulation by acting as anti-inducers. Inducer stimulates CysB protein binding to sites involved in positive regulation, and inhibits binding to the negatively autoregulated cysB promoter. For three sites with unknown function, binding is stimulated at one and inhibited at the other two.

Isorepressor of the gal regulon in Escherichia coli

Inducible overexpression of the Escherichia coli gal operon in the absence of the Gal repressor is known as ultrainduction. The requirement of induction can be eliminated by mutation of a new locus, galS, resulting in constitutive and ultrainduced levels of gal expression. Characterization of the galS gene and its product has revealed an isorepressor of the gal regulon. The Gal isorepressor is a protein of 346 amino acid residues whose amino acid sequence and cellular function, as described here, are very similar to that of Gal repressor, encoded by the galR gene. Transcription from different promoters of the gal regulon, galP1, galP2 and mglP, was examined by primer extension and reverse transcription of mRNA isolated from strains containing mutations in galR and/or galS. In strains containing a galS mutation, overexpression of gal message occurred only in the presence of inducer, while mgl message was constitutively derepressed. The galS mutation also constitutively derepressed an mglA::lacZ fusion, demonstrating that GalS is the mgl repressor. A potential operator site in the mgl promoter was identified at a position analogous to O E in gal. Thus, the gal and mgl operons constitute a regulon. Crosstalk, temporal action, induction spectrum or heteromer formation between repressor and isorepressor may help co-ordinate high affinity galactose transport and galactose utilization.

An approach for isolation of mutants defective in 35S ribosomal RNA synthesis in Saccharomyces cerevisiae.

We have developed a method to isolate mutants of Saccharomyces cerevisiae that are primarily defective in the transcription of 35S ribosomal RNA (rRNA) genes by RNA polymerase I. The method uses a system in which the 35S rRNA gene is fused to the GAL7 promoter and is transcribed by RNA polymerase II under control of the GAL regulatory system. Chromosomal mutations affecting components specifically involved in synthesis of 35S rRNA by RNA polymerase I can be suppressed by this hybrid gene in the presence of inducer (galactose) but not in its absence. We looked for mutants the growth of which depended on the presence of plasmid expressing the hybrid gene. For this purpose, we used a red/white-colony color assay as the initial screen followed by a test for galactose-dependent growth. We have thus isolated many mutants and identified at least nine genes (RRN1-RRN9) involved in 35S rRNA synthesis, two of which correspond to known RNA polymerase I subunit genes RPA190 and RPA135.

Energy transfer in lactose repressor protein modified with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate

Energy transfer between the two tryptophan residues in the lactose repressor protein and the fluorescent moiety of the cysteine-specific reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate (1,5-IAEDANS) has been examined. Modification of repressor with this compound did not affect operator or inducer binding. 1,5-IAEDANS reacted primarily with Cys140 in wild-type repressor [Schneider et al. (1984) Biochemistry 23, 2221]; in the presence of inducer, modification at Cys107 increased, while reaction at Cys140 remained unchanged. Energy transfer between tryptophans and the AEDANS moiety(ies) in wild-type lac repressor occurred with an efficiency of 6.7 +/- 1.9% in the absence and 7.8 +/- 1.6% in the presence of inducer. The distance between the Trp donor(s) and the acceptor in wild-type repressor was calculated to be in the range approximately 35 A under both conditions. The similarity in efficiency despite large differences in the amount of acceptor attached to Cys107 when inducer is bound indicates that the AEDANS group at position 107 does not participate significantly in energy transfer and that the label at position 140 acts as the primary acceptor group. The similarity of energy-transfer efficiency (7.1 +/- 3.8%) observed for 1,5-IAEDANS-modified monomeric mutant repressor (Y282D) indicates that the transfer is primarily intrasubunit in the native tetramer. Measurements using two mutant repressors (each with a single tryptophan and modified with 1,5-IAEDANS) demonstrated that both tryptophans can serve as donor in the energy-transfer process. The W201Y repressor (containing Trp220) exhibited a transfer efficiency lower than wild type (5.6 +/- 2.4%), corresponding to a slightly larger distance between the donor-acceptor pair in this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cell wall deficiency on the synthesis of polysaccharide-degrading exoenzymes: a study on mycelial and wall-less phenotypes of the fz; sg; os-1 ('slime') triple mutant of Neurospora crassa

The production of exoenzymes which degrade cellulose, polygalacturonic acid and xylan was studied in mycelial and wall-less phenotypic derivatives of Neurospora crassa obtained by vegetative selection applied to a single fz;sg;os-1 ('slime'-like) segregant (strain RCP-3) of a cross 'slime' x wild type. The unrelated stable 'slime' strain FGSC 1118 was also studied. The synthesis of polysaccharide-degrading enzymes was normally induced by polysaccharidic substrates and was sensitive to carbon-catabolite repression for both mycelium-forming phenotypes (mycelial intermediate and spheroplast-hyphal intermediate) of strain RCP-3. The stable 'slime' from RCP-3 produced cellulose-degrading activity and xylan-degrading activity constitutively but was fully sensitive to glucose repression. The stable 'slime' RCP-3 did not synthesize polygalacturonic-acid-degrading activity, even in the presence of inducers. For the stable 'slime' FGSC 1118, all of the polysaccharide-degrading activities were produced constitutively and were markedly resistant to glucose repression. The possible epigenetic origin of the different properties of stable 'slimes' RCP-3 and FGSC 1118 is considered. These results may relate to the role of the cell surface in the processing of regulatory signals which control the adaptation of the fungal cell to the nutritional environment.

Regulatory functions of the three nodD genes of Rhizobium leguminosarum biovar phaseoli

The three nodD genes of a strain of Rhizobium leguminosarum biovar phaseoli were cloned to study their effects on transcription of themselves and of the nodC genes of biovars phaseoli and viciae. Efficient transcription of nodD1 required nodD1 and was enhanced by exposure of the cells to bean exudate consistent with the presence of a nod-box preceding the noIE-nodD1 operon. Transcription of nodD2 and nodD3 was constitutive. nodC of R. leguminosarum biovar phaseoli was activated by each of the nodD genes of that biovar in the absence of inducers but expression was enhanced in cells grown with bean exudate or the flavonoids genistein or naringenin. A mutant of nodD2, lacking 60 bp at its 3' end, activated nodC in the presence of inducer, but was defective in regulating certain of the nodD genes. The nodC gene of R. leguminosarum biovar viciae responded differently to the various nodD genes of R. leguminosarum biovar phaseoli than did the nodC of the latter biovar.

The GLN3 gene product is required for transcriptional activation of allantoin system gene expression in Saccharomyces cerevisiae

We show that mutation at the GLN3 locus results in decreased steady-state levels of DAL7, DUR1,2, CAR1, and URA3 mRNAs derived from cultures grown in the presence of inducer. Basal levels of these RNA species, however, were not significantly affected by a gln3 mutation. The GLN3 product appears to affect gene expression in two ways. The pleiotropic requirement of GLN3 for induced gene expression probably derives from the need of the GLN3 product for inducer uptake into the cell and its loss in gln3 mutants. We also demonstrate that transcriptional activation, mediated by the DAL5 and DAL7 upstream activation sequences, requires a functional GLN3 gene product. This observation identified transcriptional activation as the most likely point of GLN3 participation in the expression of allantoin system genes.

Physiological studies of the regulation of beta-lactamase expression in Pseudomonas maltophilia.

The kinetics of beta-lactamase induction in Pseudomonas maltophilia IID1275/873 were investigated. Upon induction with beta-lactam antibiotics, a correlation was seen between the increase in specific beta-lactamase activity and the generation time, as well as the concentration of inducer in the medium. The specific beta-lactamase activity increased slowly within the first 0.5 generation and then more rapidly; it decreased regularly after about 2 generations of growth in the presence of inducer. This decrease could presumably be attributed to the continuous breakdown of inducer by beta-lactamases in the culture medium. In a chemostat culture with continuous supply of fresh inducer-containing medium, the specific beta-lactamase activity could be stabilized at a high level over several generations. Removal of the beta-lactam after a certain induction time showed that a short exposure of the bacteria to inducer caused induction kinetics comparable to those resulting from continuous exposure of the cells to inducer. The two beta-lactamases of P. maltophilia, L1 and L2, were induced simultaneously under various experimental conditions.

Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding.

Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.

Studies on the simultaneous production of single cell protein and polygalacturonase fromKluyveromyces fragilis

Kluyveromyces fragilis produces polygalacturonase (PG) on a lactose medium. Although the enzyme is normally repressed at high aeration levels, significant amounts of PG can be produced under such conditions when pectin is added as inducer. The productivity and yield of cell mass were not significantly affected by the presence of inducer, suggesting potential applications to current single cell protein processes from whey.

Enhanced Interleukin-1 Production by Human Monocyte Cell Lines Following Treatment With 5-Azacytidine

Recent studies suggest that DNA modification, such as methylation of specific bases or substitution of altered bases, can play an important role in the control of eukaryotic gene expression. We chose to examine the effects of two DNA modifying agents, 5-azacytidine (AZA) and 5-bromodeoxyuridine (BUdR), on interleukin-1 (IL-1) gene expression in the human monocyte cell lines THP-1 and U937.1. THP-1 produced IL-1 upon stimulation with lipopolysaccharide (LPS), whereas U937.1 did not. Following treatment with AZA, U937.1 cells could be induced to produce IL-1 beta mRNA and release IL-1, but only if a stimulus such as LPS was present. In addition, the level of IL-1 beta mRNA produced and IL-1 released by THP-1 cells after induction could be doubled by treatment with AZA. In contrast, we were unable to alter IL-1 production by treatment of cells with BUdR in the absence or presence of inducers. These results suggest that the production of IL-1 may be, in part, regulated by methylation of DNA.

Dissociation of the lactose repressor protein tetramer using high hydrostatic pressure.

Dissociation of lac repressor tetramer by high hydrostatic pressures was monitored with intrinsic tryptophan fluorescence. With the assumption of complete dissociation to monomer, tryptophan polarization data gave delta V a approximately 170 mL/mol and the concentration for 50% tetramer dissociation, C1/2, was 3.8 X 10(-8) M. Upon addition of inducer, the calculated delta V a increased to approximately 220 mL/mol and the C1/2 decreased to approximately 1 X 10(-8) M, a free energy difference of approximately 0.7 kcal. These results indicate a modest stabilization of the tetramer by the presence of inducer. Monitoring the average energy of tryptophan emission demonstrated that tetramer dissociation takes place over the same range of pressures as evidenced by the polarization data and IPTG dissociation can be more or less superimposed upon tetramer dissociation depending upon the ligand concentration used. Although the two transitions cannot be separated entirely, the delta V a for the region of the pressure dependence dominated by ligand dissociation was 69 mL/mol, an unexpectedly large value. For tetramer modified with methyl methanethiosulfonate, subunit dissociation was shifted to much higher pressures and IPTG dissociation did not occur. The delta V a for subunit association was calculated as approximately 160 mL/mol, and the C1/2 was 3.5 X 10(-9) M. Interactions at the subunit interface of the modified protein are apparently stronger than in the unmodified protein. The absence of inducer dissociation from the MMTS-modified tetramer by the application of high hydrostatic pressure suggests that the volume change for inducer binding to the modified protein is much smaller than that observed for the unmodified repressor.

Nonheritable resistance to chloramphenicol and other antibiotics induced by salicylates and other chemotactic repellents in Escherichia coli K-12.

Phenotypic resistance to chloramphenicol and ampicillin was induced in sensitive Escherichia coli K-12 strains during incubation with the following substances: acetate, acetylsalicylate (aspirin), benzoate, dimethyl sulfoxide, 1-methyl-2-pyrrolidinone, and salicylate. In addition, acetyl-salicylate and salicylate induced resistance to nalidixic acid and tetracycline. The induction of resistance was highly efficient but varied somewhat with the strain and inducer used. In the presence of inducers, from 10% to 100% of the cells formed colonies on antibiotic media, an increase of 10- to 1000-fold over the controls without inducer. After growth in the absence of these inducers, the cells were normally sensitive to the antibiotics. Thus, the resistance was not due to a heritable change. These inducers also increased the level of chloramphenicol resistance of a strain carrying cat (whose gene product inactivates chloramphenicol by acetylation). All of the inducers are chemotactic repellents for E. coli, and they are detected by the tsr gene product (with the possible exceptions of dimethyl sulfoxide and methylpyrrolidinone, whose modes of detection are not known). Nickel sulfate and cobalt sulfate, repellents that are detected by the tar gene product, neither promoted resistance to chloramphenicol nor prevented the induction of resistance by acetylsalicylate. Since several of the inducers are present in common drugs or foods, it may be of medical importance to evaluate their effects on antibiotic therapies.